ABSTRACT Fungal keratitis, primarily caused by Candida, Fusarium, or Aspergillus species, is a leading cause of blindness among corneal diseases. Amphotericin B (AmB) remains the gold standard in the treatment of Candida keratitis and is also the fallback option for other corneal fungal infections. Although widely used off-label in ocular infections, ophthalmic AmB formulations are not available commercially. The compounded formulations used - reconstituted AmB for injection - are not optimized for ocular administration and suffer from several deficiencies (e.g. stability of reconstituted solution, anionic surfactant concentrations) that limit effective use of AmB in fungal infections of the eye. This application is based on the hypothesis that novel AmB loaded nanostructured lipid carriers, using castor oil as the liquid lipid and modified with PEG of specific molecular weights (PEG-NLC-AmB), will provide a stable, long acting and effective multidose ophthalmic formulation. The hypothesis is strongly supported by the preliminary data that demonstrates that these specific PEG-NLC-AmB formulations are stable on reconstitution, autoclavable, and more potent in vitro against Candida albicans and Aspergillus fumigatus compared to unPEGylated NLCs and AmBisome and is equivalent to that of (if not better than) Fungizone. Moreover, addition of a preservative, benzalkonium chloride (BAC), to the PEG-NLC-AmB formulation did not cause any apparent change in the particle size, polydispersity index or in vitro activity, whereas, agglomeration/precipitation and inconsistent in vitro activity was observed with the two reconstituted commercial formulations in the presence of BAC. Furthermore, ocular bioavailability of AmB from PEG-NLC-AMB formulations, following topical instillation, was equivalent to that obtained with AmBisome. The objective of this Phase I application is to optimize the promising formulation leads (including surface modified and in situ gelling formulations) and to evaluate in vivo efficacy. The project goals will be met through two specific aims: Under Aim 1 the PEG-NLC-AmB lead formulations will be optimized with respect to process and storage stability (lyophilized as well as post reconstitution), formulation characteristics, preservative compatibility, corneal permeability, cytotoxicity and in vitro fungicidal activity. The best two formulations will proceed to Aim 2 to test the efficacy against Candida keratitis in the New Zealand white rabbits. Compounded AmB formulations will serve as the control formulation. Only one eye of each rabbit will be infected but the formulation will be administered to both eyes. Efficacy will be evaluated at two doses, 0.5 and 1.0 mg/mL, of each of the formulations. The infected eye of each rabbit will be examined, and clinical disease severity scores will be assigned. The uninfected eye will be assessed for irritation using the Draize numerical scoring system to assess safety of the formulations. All animal studies will be undertaken under approved protocols. Successful development of the PEG-NLC-AmB ophthalmic formulation will provide a much-needed affordable, effective and safe option for treating and managing fungal keratitis and other ocular (uveal/retinal), mucosal, topical and/or systemic fungal infections.